DNA rearrangements affect genome stabilities in most organisms, and cause major diseases including cancer in humans. They also occur as programmed processes that play specific roles in cell differentiation in many organisms. One unusual class of DNA rearrangements occurs genome-wide to alter overall structures of chromosomes. Although known for more than a century, their molecular nature is poorly understood. This proposal will examine this type of DNA rearrangements in the model eukaryote Tetrahymena thermophila. Past studies of this project have shown that the somatic genome of this organism is dramatically re-structured during development through two processes: chromosome breakage and DNA deletion. DNA deletion occurs at several thousand sites to excise specific fragments of diverse sizes and sequences, which together comprise about 15% of the genome. Recognition for DNA deletion involves two cis-acting sequences: flanking regulatory sequences and internal promoting sequences. This proposal will determine how these sequences are recognized and how they interact with each other by testing transgenic strains with specific alterations in these sequences, and test the idea that DNA deletion serves as a surveillance mechanism to remove invading genetic elements such as transposons. Although not coding for proteins, these sequences are transcribed during nuclear differentiation to produce double stranded RNA. This study will determine how this transcription may regulate DNA deletion. The internal promoting sequences will inhibit DNA deletion if they are artificially placed in the old somatic nucleus. This inhibition perpetuates itself in the following generations and becomes an epigenetic trait. The molecular basis of this inhibition and its relationship to the unusual transcription will be examined. Chromosome breakage occurs at approximately 200 specific sites marked by a 15 bp signal. It cleaves DNA and adds new telomeric sequences to the free ends. These minichromosomes are maintained indefinitely in the somatic nucleus during vegetative growth. This proposal will search for genetic mutants and identify genes that control this process using a special transgenic strain for the screen. It will also determine the possibility that a recently discovered homing endonuclease gene, F-Tthl, encodes the enzyme that breaks chromosomes. Finally, the proposal will research for new genes involved in DNA rearrangements by using an approach that identifies genes based on the location of their encoded proteins. By combining these approaches, the study hope to reveal the underlying molecular mechanisms of these processes, uncover their relationships to other cellular processes such as chromosome condensation and DNA repairs and gain insights to their biological roles.